Method and means for detecting inflammatory processes

ABSTRACT

A method of detecting inflammatory processes comprises (a) administering per-orally or per-rectally a composition comprising a diagnostically effective amount of L-[guanido- 15 N 2 ]-arginine, L-[guanido- 15 N]-arginine, their mixtures and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier; (b) collecting a sample of exhaled air, saliva or urine; (c) determining  13 NO in the air sample or its transformation products  15 IN-nitrite and/or  15 N-nitrate in the saliva sample or urine. Also disclosed is a corresponding diagnostic composition for use in diagnosing inflammatory processes and a method for its manufacture.

This is a 371 of PCT/SE00/02054, filed Oct. 24, 2000.

FIELD OF THE INVENTION

The present invention relates to a method for determining nitrogen oxideformed in inflammatory processes, to a means for carrying out the methodand to a method of manufacture of the means.

BACKGROUND OF THE INVENTION

Nitrogen oxide (NO) has important biological functions. It is thestructurally simplest mediator in the human body as well as one of themost important weapons of its immune defense. In its later function itis excreted by activated macrophages to kill foreign microorganisms andcells recognized as foreign. In this second capability nitrogen oxidemay be considered an inflammation marker and has been recognized as suchin, for instance, inflammatory processes in the gastro-intestinal tract,such as ulcerative colitis and Crohn's disease and celiac disease. Inthe lung it may assume both roles. It is recognized that levels ofnitrogen oxide excretion are raised in asthmatics. Thus it may beconsidered a marker for asthma which also comprises an importantinflammatory component.

In the body nitrogen oxide is formed from L-arginine by hydroxylation ofone of the guanidino nitrogens in a reaction catalyzed by one of theisoforms of the enzyme nitrogen oxide synthase (NOS). In a complexreaction the thus formed hydroxyimino intermediate is oxidatively splitinto nitrogen oxide and L-citrulline.

Nitrogen oxide may be sampled in situ and measured by, for instance,chemoluminescense (WO 96/17244; WO 97/37587). In situ sampling, forinstance in the gastrointestinal tract and in the urinary tract, oftenis difficult or at least time-consuming. Samples of exhaled air maycontain nitrogen oxide formed in the pulmonary system but also formedelsewhere because of the considerable solubility of NO in water andlipids which makes it freely diffusible in the body. Increased levels ofNO in exhaled air thus may be due to inflammation in the pulmonarysystem as well as elsewhere. This detracts from the potential usefulnessof nitrogen oxide as an inflammation marker.

OBJECTS OF THE INVENTION

It is an object of the invention to provide an improved method fordetermining nitrogen oxide formed in the body.

It is another object of the invention to provide a means for carryingout said method.

Further objects of the invention will be apparent from the followingdescription of the invention and preferred embodiments thereof, and fromthe appended claims.

SUMMARY OF THE INVENTION

According to the present invention is provided a method of theaforementioned kind comprising the per-oral or perrectal administrationof a composition comprising a diagnostically effective amount ofarginine terminally labeled with the stable nitrogen isotope ¹⁵N fordetermination, directly or indirectly, of ¹⁵NO in exhaled air or saliva.It is more preferred for both terminal nitrogen atoms to be labeled. Inthis specification is understood by ‘terminally labeled’ the labeling ofone or both of the terminal guanido nitrogen atoms of L-arginine. Directdetermination of ¹⁵NO implies that the compound is measured as such,whereas indirect determination implies that a product into which it hasbeen transformed is measured such as, for instance, ¹⁵N-nitrite or¹⁵N-nitrate.

According to a first preferred aspect of the invention is provided amethod for per-oral administration of a diagnostic compositioncomprising a diagnostically effective amount of L-arginine terminallylabeled with the stable nitrogen isotope ¹⁵N for release in the smallintestine and/or in the upper part of the large intestine.

According to a second preferred aspect of the invention is provided amethod of the aforementioned kind comprising the per-rectaladministration of a composition comprising a diagnostically effectiveamount of arginine terminally labeled with the stable nitrogen isotope¹⁵N for release in the lower part of the large intestine.

According to a third preferred aspect of the invention is provided amethod of the aforementioned kind comprising the per-oral administrationof a diagnostic composition comprising a diagnostically effective amountof L-arginine terminally labeled with the stable nitrogen isotope ¹⁵Nfor inhalation.

Preferred assaying methods for ¹⁵N comprise: IR-spectrometry, laserspectrometry, gas chromatography and mass spectrometry, and theircombinations. The determination of ¹⁵N as NO by any of these methodsmust take into account the fact that atmospheric nitrogen is a mixtureof ¹⁴N (99.63%; isotopic abundance) and ¹⁵N (0.37%). In a sample ofexhaled air is thus the amount of ¹⁵N in excess of the natural isotopicabundance of ¹⁵N that is representative of labeled nitrogen in arginine.The determination of ¹⁵N as NO by any of these methods must also takeinto account the purity of the label. The fact that ¹⁴N and ¹⁵N differin their mass by 6.6%, in consideration of the natural abundance of ¹⁶Ooxygen being 99.76%, makes ¹⁵N particularly attractive as a marker. Itis thus ¹⁵N¹⁶O which is determined at m/e=31 in the presence of ¹⁴N₂(m/e=28), ¹⁴N¹⁵N (m/e=29), ¹⁴N¹⁶O (m/e=30) formed from non-labeledL-arginine, ¹⁶O₂ (m/e=32), ¹⁴N¹⁸O (m/e=32), ¹⁶O¹⁷O (m/e=33), ¹⁶O¹⁸O(m/e=34). Because of the low natural abundance of ¹⁷O (0.037%) ¹⁴N¹⁷O(m/e=31) will be present in minute amounts only, and can be disregardedfrom.

It is advantageous to partially or fully separate the components of agaseous sample containing ¹⁵NO in a gas chromatograph before injecting asample of the fraction containing nitrogen oxide in the massspectrometer. Methods for such separation are well known in the art.

Since NO formed from ¹⁵N₂-arginine is isotopically quantitativelydifferent from NO formed from unlabeled arginine, the amount of NOformed at or near the site where the labeled arginine is administeredcan be determined.

Thus, according to a further preferred aspect of the invention, the siteof administration of terminally ¹⁵N-labeled arginine is different fromthe site of detection of ¹⁵N-labeled NO formed therefrom. For instance,terminally labeled ¹⁵N-arginine can be administered orally or rectallyto a person suspected to suffer from ulcerative colitis or Crohn'sdisease, and the intestinally formed ¹⁵N-labeled NO be determined in theexhaled air or, in the form of nitrite or nitrate, in saliva.

According to still another preferred aspect of the invention labelednitrogen oxide formed from correspondingly terminally labeled¹⁵N-arginine can be assayed by measurement of one or several of theproducts to which it is biologically transformed, in particular nitrate.It is known that a substantial amount of nitrogen oxide entering thebloodstream is oxidized to nitrate in which form it is excreted by thekidneys. It is thus within the scope of the invention to determine theformation of labeled nitrogen oxide by measuring labeled ¹⁵NO₃ ⁻inurine. Another important path for excretion is from the salivary glands.It is thus also within the scope of the invention to determine theformation of labeled nitrogen oxide from correspondingly ¹⁵N-labeledL-arginine by measuring ¹⁵N-labeled NO₃ ⁻in saliva and/or by measuring¹⁵N-labeled NO₂ ⁻in saliva to which nitrate is rapidly transformed bythe action of certain bacteria colonizing the surface of the tongue.

According to still another preferred aspect of the invention ¹⁵N-labeledarginine can be administered to the lungs in form of a spray or mist forthe detection of inflammatory processes in the respiratory system, inparticular of asthma, nasal inflammation, and sinusitis.

According to still another preferred aspect of the invention ¹⁵N-labeledarginine can be administered to the circulating blood in form of aninjection or infusion for the detection of inflammatory processes in theblood (sepsis) or in tissues in contact with circulating blood.

According to the present invention is also disclosed a diagnosticcomposition for use in diagnosing inflammatory processes, comprising adiagnostically effective amount of terminally labeled ¹⁵N-arginine and apharmaceutically acceptable carrier.

Also disclosed is a process for the manufacture of such compositioncomprising formulating a diagnostically effective amount of terminallylabeled ¹⁵N-arginine and a pharmaceutically acceptable carrier into apharmaceutical composition for per-oral or per-rectal administration.

It is preferred for the compositions according to the invention forper-oral administration to be selected from the group consisting of:enteric delayed release compositions for per-oral administrationreleasing ¹⁵N-arginine in the small intestine and/or the upper part ofthe large intestine; nebulizable aqueous solutions of and powderscontaining terminally labeled ¹⁵N-arginine for administration to thebronchi and the lung.

It is preferred for the compositions according to the invention forper-rectal administration to be selected from enemas, foams, andsuppositories.

DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1 Terminally N-labeledArginine

L-[guanido-¹⁵N₂]-arginine is commercially available from ICNPharmaceuticals, Inc. (Costa Mesa, Calif.) and Tracer Technology, Inc.(Somerville, Mass.). Fully ¹⁵N-labeled arginine can also be used. It canbe produced by Corynebacterium herculis or Brevibacterium flavum strainscarrying the recombinant DNA pCarg11 or pCarg110 according to the methoddisclosed in U.S. Pat. No. 5,017,482, which is hereby incorporated byreference, with the proviso that ¹⁵N₃ ammonium sulphate is substitutedfor ammonium sulphate as the nitrogen source. L-[guanido-¹⁵N₂]-argininecan be used in form of the free base or a pharmaceutically acceptablesalt, such as the hydrochloride or aspartate.

EXAMPLE 2 Enteric Tablet

An enteric tablet for release in the small intestine and the upper largeintestine can be prepared according to EP 0 502 092 A1 by substituting aterminally N-labeled arginine for any of the glucocorticoids disclosedtherein. A suitable amount of terminally N-labeled arginine, such asL-[guanido-¹⁵N₂]-arginine, is 50 mg, in the form of the free base or apharmaceutically acceptable salt thereof, such as the hydrochloride.

EXAMPLE 3 Suppository

A suitable type of suppository can be made by use of the Salazopyrin®EN, Pharmacia & Upjohn, enema composition in which the active principlesulfasalazine (500 mg) is exchanged for 50 mg L-[guanido-¹⁵N₂]-arginineand 450 mg of suitable neutral constitutents, such as calcium carbonate.

EXAMPLE 4 Solution for Inhalation Spray

100 mg L-[guanido-¹⁵N₂]-arginine are dissolved in 10 ml of sterilewater. The solution is administered by a nebulizer capable of dispensingmeasured doses.

EXAMPLE 5 Determination of Inflammatory Conditions in the SmallIntestine and the Upper Part of the Large Intestine

An enteric tablet of EXAMPLE 2 is administered to the person suspectedof inflammation in a fasting state. Breath samples (100 ccm) are takenin 10 min intervals starting at time of administration.

EXAMPLE 6 Determination of Inflammatory Conditions in the Lower Part ofthe Large Intestine

A suppository of EXAMPLE 3 is per-rectally administered to the personsuspected of inflammation being in a fasting state. Breath samples (100ccm) are taken in 10 min intervals starting at time of administration.

EXAMPLE 7 Determination of Inflammatory Conditions in the Lung

A metered dose (1 ccm) of the solution for inhalation of EXAMPLE 4 isnebulized for inhalation by the patient using of a state-of-the-artnebulizer. Breath samples (100 ccm) are taken in 5 min intervalsstarting at time of administration.

EXAMPLE 7 Determination of Inflammatory Conditions in the Blood

An intravenous infusion of L-[guanido-¹⁵N₂]-arginine is administered toa person suspected of sepsis. Breath samples (100 ccm) are taken in 10min intervals starting at time of administration.

EXAMPLE 8 Determination of ¹⁵NO in Gaseous Samples

See: D C Macallan et al., Am. J. Physiol. 272 (6, part 2), p.R1888–R1896 (1997).

EXAMPLE 9 Determination of Urinary ¹⁵NO₃ ⁻

See: P Forte et al., Measurement of Nitric Oxide Synthesis in HumansUsing L-[¹⁵N₂]Arginine. Methods in Enzymology 301 (1999) 92–98. Themethod is easily modified for measurement of nitrate in saliva.

1. A method of detecting the existence of an inflammatory processcomprising: administering by inhalation to a person a compositioncomprising a diagnostically effective amount ofL-[guanido-¹⁵N₂]-arginine, L-[guanido¹⁵N]-arginine, their mixtures andpharmaceutically acceptable salts thereof, and a pharmaceuticallyacceptable carrier, collecting a sample of exhaled air, saliva or urine,determining ¹⁵NO in the air sample or its transformation products¹⁵N-nitrite and/or ¹⁵N-nitrate in the saliva sample or urine, whereinthe inflammatory process is asthma or a bacterial or viral infection ofthe respiratory system.
 2. The method of claim 1, wherein thecomposition administered is a spray or mist for inhalation from anaqueous solution of L-[guanido-¹⁵N₂]-arginine, L-[guanido-¹⁵N]-arginine,their mixtures, and pharmaceutically acceptable salts thereof.
 3. Themethod of claim 1, wherein the composition administered is a inhalationsuspension in air from a powder comprising L-[guanido-¹⁵N₂]-arginine,L-[guanido-¹⁵N]-arginine, their mixtures and pharmaceutically acceptablesalts thereof.
 4. The method of claim 1, wherein said amount is from 1mg to 2,000 mg.
 5. The method of claim 1, wherein the sample collectedis exhaled air or saliva.
 6. The method of claim 1, whereindetermination is by IR-spectrometry, laser spectrometry, massspectrometry, gas chromatography, and their combinations.
 7. The methodof claim 6, wherein the sample collected is exhaled air or saliva. 8.The method of claim 7, wherein said amount is from 1 mg to 2,000 mg. 9.The method of claim 8, wherein the composition administered is a sprayor mist for inhalation from an aqueous solution ofL-[guanido-¹⁵N₂]-arginine, L-[guanido-¹⁵N]-arginine, their mixtures, andpharmaceutically acceptable salts thereof.
 10. The method of claim 8,wherein the composition administered is a inhalation suspension in airfrom a powder comprising L-[guanido-¹⁵N₂]-arginine,L-[guanido-¹⁵N]-arginine, their mixtures and pharmaceutically acceptablesalts thereof.